====== Engineering the Human Gut Microbiome ====== ** 16th of November - David Kong (MIT) ** [[http://bio.academany.org/files/HTGAA_Kong.pdf|David Kong's slides]] [[http://bio.academany.org/files/HTGAA_Kearney.pdf|Sean Kearney's slides]] [[http://connectmedia.waag.org/AcadeMany/bio-2016/11/16/index.html|recordings]] {{vimeo>191810213?medium}} {{vimeo>191829516?medium}} The human gut microbiota is one of the most densely populated ecosystems of microorganisms on earth. With an estimated 100 trillion microorganisms, the gut is an extraordinarily complex system of microbe-microbe and microbe-host interactions. A growing body of research is beginning to elucidate the diverse impacts the gut microbiota plays in human health and development, from nutrition, to disease, and even cognition. Recently, with the success of fecal matter transplants (FMTs) to treat infectious disease, microbes are emerging as a unique therapeutic. Model systems to both prototype and study complex polymicrobial systems are a necessity for producing robust microbial communities that can be engineered at both the genetic level (subcellular) and population level (multicellular). David Sun Kong, Ph.D. ====== Readings ====== 'Some of My Best Friends Are Germs,' by Michael Pollan, NYTimes: http://www.nytimes.com/2013/05/19/magazine/say-hello-to-the-100-trillion-bacteria-that-make-up-your-microbiome.html?_r=0 'Microbiota-Targeted Therapies: An Ecological Perspective' by Katherine P. Lemon, Gary C. Armitage, David A. Relman, and Michael A. Fischbach http://stm.sciencemag.org/content/4/137/137rv5.full-text.pdf+html 'Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow' by Hyun Jung Kim, Dongeun Huh, Geraldine Hamilton and Donald E. Ingber http://pubs.rsc.org/en/content/articlehtml/2012/lc/c2lc40074j ====== Homework ====== ---------------------------------------------------------------------- **(1) 3D print a 14 mL culture tube in at least one material. Culture a bacterial strain of your choice in this tube and compare the growth rate (optical density) over time versus a polystyrene control tube.** Ideally use a strain featuring antibiotic resistance and culture in the presence of an antibiotic. Tube and cap design files: http://metafluidics.com/devices/14-ml-culture-tube/ __Extra credit:__ Culture multiple combinations of tube materials and strains, comparing growth rates for each against polystyrene. {{::screen_shot_2015-11-11_at_12.22.45_am.png?200|}} ---------------------------------------------------------------------- ** (2a) Design a milli- or micro-fluidic 'artificial gut' or other 'organ-on-a-chip' device to be utilized, at a minimum, for cell culture.** Feel free to design your device in 2D-CAD software or vector drawing tool (e.g. Adobe Illustrator, AutoCAD) or 3D design tool (e.g. Rhino, SolidWorks). Rhino (for Mac): https://www.rhino3d.com/ Example designs: http://metafluidics.com/devices/x-mixer/ http://metafluidics.com/devices/air-pocket-generator-droplet-generator/ ---------------------------------------------------------------------- ** (2b) Fabricate your device, or at least one component of your device.** Document the following aspects of fabrication and function in your wiki: * What features of your organ are you attempting to emulate? * How is your device intended to function? * Were you able to fabricate your device? Which components? Which parts 'worked' and which ones didn't? * What will you aim to improve for your next iteration of design + build? Please include photos / screen shots of your digital designs, fabrication process, and final structures! An example protocol for fabricating an 'organ-on-a-chip': http://www.seas.upenn.edu/~biolines/publications/NatProtoc-13-Huh-protocol.pdf ---------------------------------------------------------------------- ** (2c) Culture the organism from (1) in your milli- or micro-fluidic device.** Run a negative control in a device with liquid media only. Collect the liquid culture from your device (+/- bacteria) and plate in the presence of an antibiotic. Report colonies for the +/- experiments. ---------------------------------------------------------------------- ** (3) Share your 'final' device designs on 'Metafluidics'** (www.metafluidics.com), including Bill of Materials, assembly instructions, and any associated hardware. Irrespective of how far you get in (2), please share your latest iteration! You can always update your device later. Login: metafluidics | PW: bocoup ---------------------------------------------------------------------- **Materials** * At least one bacterial strain of your choice (ideally resistant to an antibiotic). * Liquid media suitable for growing that strain. * Antibiotic * 14 mL polystyrene culture tubes * Tool for measuring optical density (spectrophotometer) * Petri dishes for plating If you do not have access to a spectrophotometer, you can use 'MacFarland Turbidity standards' to indirectly measure optical density. See: http://biobuilder.org/protocol-a/ Click 'Day 4'. Also: http://biobuilder.org/wp-content/uploads/2013/11/MiE_00012.pdf Starting bottom of page 267. * 3D printer of any kind or use of a service (e.g. Shapeways) * Tubing to interface with your fluidic devices * Syringes to interface with tubing for fluid handling ---------------------------------------------------------------------- **Cell Culture in 3D-printed Tube Example** __Growth Conditions__ An overnight culture of Escherichia coli was inoculated with a single colony in 5 mL of sterile LB Broth + 50 mg/mL kanamycin and shaken at 37°C. Prior to use, each printed tube was UV-irradiated for 15 minutes. After irradiation, 5mL of sterile LB Broth + 50 mg/mL kanamycin was inoculated with 100 μL of the overnight culture and considered time 0. Tubes were incubated in a 37°C shaking incubator between OD600 measurements. __Optical Density Measurements__ An Eppendorf BioPhotometer (Eppendorf, Hamburg, Germany) was used with 8.5 mm centre height uVettes (Eppendorf, Cat. no.: 952010069) with a 100 μL sample volume. Measurements were made at 0, 2, 4, 6, 8 and 24 hours. Samples were diluted 1:5 in media at 6, 8, and 24 hours. All samples were blanked against media incubated in a tube of the given material.